Natural Killer Cells Do Not Attenuate a Mouse-Adapted SARS-CoV-2-Induced Disease in Rag2-/- Mice

Abstract

This study investigates the roles of T, B, and Natural Killer (NK) cells in the pathogenesis of severe COVID-19, utilizing mouse-adapted SARS-CoV-2-MA30 (MA30). To evaluate this MA30 mouse model, we characterized MA30-infected C57BL/6 mice (B6) and compared them with SARS-CoV-2-WA1 (an original SARS-CoV-2 strain) infected K18-human ACE2 (K18-hACE2) mice. We found that the infected B6 mice developed severe peribronchial inflammation and rapid severe pulmonary edema, but less lung interstitial inflammation than the infected K18-hACE2 mice. These pathological findings recapitulate some pathological changes seen in severe COVID-19 patients. Using this MA30-infected mouse model, we further demonstrate that T and/or B cells are essential in mounting an effective immune response against SARS-CoV-2. This was evident as Rag2^-/- showed heightened vulnerability to infection and inhibited viral clearance. Conversely, the depletion of NK cells did not significantly alter the disease course in Rag2^-/- mice, underscoring the minimal role of NK cells in the acute phase of MA30-induced disease. Together, our results indicate that T and/or B cells, but not NK cells, mitigate MA30-induced disease in mice and the infected mouse model can be used for dissecting the pathogenesis and immunology of severe COVID-19.

Keywords: B cells; COVID-19; MA30; NK cells; SARS-CoV-2; T cells.

Figure 1

Dose-dependent effects of MA30 infection in B6 mice: (A) Body weight changes in 8-week-old mice infected with medium (Med) and low (Low) doses (n = 4 mice/group). p = 0.0248, comparing the body weight (BW) changes between the groups by two-way ANOVA analysis. (B) Viral subgenomic N RNA in the infected 8-week-old mice (from A) was measured using qPCR. (C) Body weight changes after the infection. ** p = 0.0051 (n = 5 per group) derived from comparing low- vs. medium-dose groups by mixed-effects analysis. (D) Survival rates over 10 days post infection (DPI) for each group (n = 5/group). The log-rank (Mantel-Cox) test showed a p-value of 0.0104. (E) Viral subgenomic N RNA was quantified through qPCR. The y-axis represents normalized values (log₁₀ copies per 100 ng). Twelve-week-old B6 mice (shown in C–E) were divided into three groups and intranasally inoculated with three different doses of CoV2-MA30: high (2 × 10⁵ TCID₅₀), medium (5 × 10⁴ TCID₅₀) (Med), and low (1 × 10⁴ TCID₅₀). Data from the high-dose group were derived from an independent experiment. (F) Body weight differences between the 8-week-old and 12-week-old mice infected with both medium and low doses of MA30. **** p < 0.0001 was analyzed by mixed-effects analysis for the left panel or two-way ANOVA analyses for the right panel (n = 4/group). * p < 0.05 was analyzed by using Sidak’s multiple comparisons.

Figure 2

Viral tropisms and lung pathological changes in MA30 infected B6 and CoV2-infected K18-hACE2^+/− mice. Twenty-week-old B6 and K18-hACE2 mice shown in (A–E) were intranasally infected with CoV2 MA30 (5 × 10⁴ TCID₅₀) and CoV2 MA1 (1 × 10⁴ TCID₅₀), respectively. (A,B) Epithelial cell infection of the nasal turbinates in the infected B6 (A) and K18-hACE2 mice (B) at 3 DPI. (C,D) Epithelial cell infection in the bronchus and bronchioles of CoV-2 MA30-infected B6 mice at 3 DPI (C) and in the alveoli’s epithelial cells of the WA1-infected K18-hACE2 mice at DPI (D). Cross-sections of the nasal turbinate and lung from a 20-week-old B6 mouse and a K18-hACE2 mouse. Immunostaining with the anti-cytokeratin marker in red and the anti-SARS-CoV-2 N protein in green. (E) Viral tropism was assessed by quantifying infected cells within the bronchial epithelium (left panel, E) and lung alveoli (right panel, E) of the infected mice (n = 3 mice per group). Viral infection was significant in the bronchial epithelium but not the alveoli, as indicated by unpaired t-test (p = 0.0100 and 0.1040, respectively). (F) Extensive edema in the lungs of infected B6 mice via hematoxylin and eosin (H/E) staining. (G) Extensive inflammation seen in the infected K18-hACE2 mice. (H) Quantitative analysis of lung histological changes in the infected mice (n = 9 B6 at 3 DPI, n = 3 K18-hACE2 at 3 DPI, n = 6 B6 at 5–6 DPI, n = 12 at 5–6 DPI). Mouse lung tissues shown in (F–G) were collected from multiple experiments with K18-hACE2 male and female mice infected with 2.0 × 10⁵ PFU/mouse, and B6 mice infected with the high dose of 5 × 10⁴ TCID₅₀. Pattern recognition algorithms were used to quantify the percentage of lung area affected by inflammation or edema. * p < 0.05.

Figure 3

Rag2 deficiency accelerates body weight loss and sustains viral load. B6 and Rag2^−/− 8-week-old mice were intranasally infected with a medium (Med) and low (Low) dose of CoV2-MA30 and monitored for 15 DPI (B6 Med, n = 4; Rag2^−/− med, n = 4; B6 Low, n = 5; Rag2^−/− low, n = 4). (A) Body weight changes in the infected Rag2^−/− and B6 mice. p < 0.0001, comparing BW combined by medium- and low-dose groups by three-way ANOVA. (B) Viral subgenomic RNA in the mice. Viral subgenomic N RNA was measured through qPCR, and significant difference was found between Rag2^−/− and B6 groups in both medium- (p = 0.0002) and low-dose (p < 0.0001) groups, using unpaired t-test. (C) Viral subgenomic N RNA detection in multiple extra-pulmonary tissues, including the brain, spleen, and kidney. (D,E) Viral infection detected via viral S protein staining. Anti-SARS S protein antibody in green. Representative images of viral S staining of B6 and Rag2^−/− mice infected with the medium dose of CoV2-MA30 at DPI 15. (E) Quantification of viral S protein positive cells in the lungs of all medium-dose and some low-dose mice with p-value 0.0019 from two-tailed unpaired t-test (n = 7 B6, n = 5 Rag2^−/−). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Transcriptomic analysis of the lungs of the infected Rag2^−/− and B6 (Rag2^+/+) mice: Bulk RNA seq analyses were conducted on the lungs of the infected 14-week-old Rag2^−/− (n = 3) and B6 males (n = 3) at 3 days post-infection (DPI) by MA30 (5 × 10⁴ TCID₅₀). Differentially expressed genes (DEGs) were generated by Cuffdiff and EdgeR (p < 0.05). (A) Dot plot of changed pathways in the lungs of Rag2^−/− mice as compared to B6 mice. EdgeR DEGs (Table S1) were used for ShinyGo 0.80 gene enrichment analysis (need to cite: ShinyGO: a graphical gene-set enrichment tool for animals and plants). (B) Volcano plot showing fold-change and p-value of DEGs between Rag2^−/− mice and B6 mice generated by Cuffdiff (Table S2). Key genes involved in primary immunodeficiency pathway, B cell and T cell receptor signaling pathways, Th1, Th2, and Th17 cell differentiation pathways, and interferon response genes are indicated with black line.

Figure 5

The depletion of NK cells in Rag2^−/− mice did not impact MA30-induced diseases. Eighteen-week-old male Rag2^−/− mice were infected with medium dose of MA30 (2 × 10⁴ TCID₅₀) and treated with either anti-NK or the isotype control (n = 5/group). (A) Body weight (BW) changes in the mice. The infected mice were monitored for 8 DPI and no difference was found using two-way ANOVA. (B) Survival rate in the mice. No difference was found in the mice using log-rank (Mantel-Cox) test. (C) Viral subgenomic N RNA analysis. No difference was found in viral subgenomic N RNA of the mice. (D) Lung histological changes. Representative images show comparable edema between anti-NK1.1 treated and control groups. (E) Quantitative analysis of edema. There is no significant difference found between them using unpaired t-test.